Enzymes for improved biomass conversion

ABSTRACT

Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/671,208, filed Jul. 13, 2012, the contents of which are incorporatedby reference in their entirety.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under ContractNo. DE-AC36-08GO28308 between the United States Department of Energy andAlliance for Sustainable Energy, LLC, the Manager and Operator of theNational Renewable Energy Laboratory.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file entitled “12-28_ST25.txt,” having a size in bytes of 64 kb andcreated on Jul. 12, 2013. Pursuant to 37 CFR §1.52(e)(5), theinformation contained in the above electronic file is herebyincorporated by reference in its entirety.

BACKGROUND

Lignocellulosic biomass is an abundant source of fermentable sugars, andbiofuels derived from these renewable sources represent one of the bestalternatives to petroleum-based fuels. Efficient conversion oflignocellulosic biomass, however, remains a challenge due to itsinherent recalcitrance. Given the current state of technology ofsimultaneous saccharification and fermentation (SFF) and the commercialenzyme cocktails available, various chemical and thermal pretreatmentsteps are required to achieve meaningful conversation of biomass.Another alternative seen as viable and cost competitive for the futureis consolidated bioprocessing (CBP), in which case saccharolytic enzymeare produced by the CBP organisms, which also ferment sugars releasedfrom biomass to the end product.

In nature, most cellulolytic organisms are of two types: those withnon-complexed cellulases, xylanases, and hemicellulases produced byaerobic fungi and most bacteria; and those where cellulases, xylanases,and hemicellulases are complexed on a protein scaffold. This latter caseis known only for a few anaerobic bacteria and fungi. In both cases, theenzymes secreted have a wide range of complexity but are mostly equippedwith a single catalytic domain. An alternate enzymatic system, midwaybetween the two previous paradigms, is one where the most abundantenzymes secreted are not only multi-modular but possess more than onecatalytic domain. This strategy could present several advantages; itallows the synergistic effects between several catalytic domains usuallyfound in cellulosomal systems but also lessen problems that cellulosomesmay encounter due to their size.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods that aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

Exemplary embodiments provide methods for degrading cellulose orlignocellulosic biomass by contacting a cellulose containing material orlignocellulosic biomass with an enzyme cocktail comprising athermostable enzyme comprising a GH9 domain and a GH48 domain and athermostable β-glucosidase.

In certain embodiments, the thermostable enzyme comprising a GH9 domainand a GH48 domain is from a bacterium of the genus Caldicellulosiruptor,such as Caldicellulosiruptor bescii CelA.

In some embodiments, the thermostable β-glucosidase is from a bacteriumof the genus Thermotoga, such as Thermotoga maritima.

In further embodiments, the enzyme cocktail further comprises athermostable endoglucanase, which may be from a bacterium of the genusAcidothermus, such as Acidothermus cellulolyticus. One example isAcidothermus cellulolyticus E1.

Also provided are enzyme cocktails comprising Caldicellulosiruptorbescii CelA, a thermostable β-glucosidase, such as a β-glucosidase fromthe bacterium Thermotoga maritima, and a thermostable endoglucanase,such as Acidothermus cellulolyticus E1.

Further provided are methods for producing a biofuel fromlignocellulosic biomass by contacting the lignocellulosic biomass withan enzyme cocktail described herein and converting the sugars to abiofuel by fermentation.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than limiting.

FIG. 1 shows the nucleotide (A; SEQ ID NO:1) and amino acid (B; SEQ IDNO:2) sequences for CelA from Caldicellulosiruptor bescii. Locations ofthe GH9 and GH48 domains are indicated in bold and underline,respectively.

FIG. 2 shows the nucleotide (A; SEQ ID NO:3) and amino acid (B; SEQ IDNO:4) sequences for β-glucosidase from Thermotoga maritima.

FIG. 3 shows the nucleotide (A; SEQ ID NO:5) and amino acid (B; SEQ IDNO:6) sequences for E1 from A. cellulolyticus.

FIG. 4 shows the avicel conversion by CelA at three differenttemperatures compared to CBH1/E1 at the same loading (15 mg/g).

FIG. 5 shows the glucan conversion by CelA and C. bescii culture brothon switchgrass substrates (A) or corn stover substrates (B) subjected tovarious pretreatments.

FIG. 6 shows the conversion of xylan present in native switchgrass byCelA.

FIG. 7 shows a comparison of avicel conversion by CelA alone or incombination with β-glucosidase. Loadings were 15 mg/g CelA and 14 mg/gCelA 1 mg/g+β-glucosidase, respectively.

FIG. 8 shows a comparison of dilute acid pretreated corn stoverconversion by CelA alone or in combination with β-glucosidase. Loadingswere 15 mg/g CelA and 14 mg/g CelA+1 mg/g β-glucosidase, respectively.

FIG. 9 shows a comparison of avicel conversion by CelA alone or incombination with E1. Loadings were 15 mg/g CelA and 11 mg/g CelA 4mg/g+E1, respectively.

FIG. 10 shows a comparison of dilute acid pretreated corn stoverconversion by CelA alone or in combination with E1. Loadings were 15mg/g CelA and 11 mg/g CelA 4 mg/g+E1, respectively.

FIG. 11 shows a comparison of avicel conversion by CelA alone or incombination with β-glucosidase and E1. Loadings were 15 mg/g CelA and 10mg/g CelA 1 mg/g+β-glucosidase+4 mg/g E1, respectively.

FIG. 12 shows a comparison of dilute acid pretreated corn stoverconversion by CelA alone or in combination with β-glucosidase and E1.Loadings were 15 mg/g CelA and 10 mg/g CelA 1 mg/g+β-glucosidase+4 mg/gE1, respectively.

FIG. 13 shows a comparison of de-acetylated, dilute acid pretreated cornstover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T.maratima β-glucosidase) or a commercial enzyme preparation (Ctec2).

FIG. 14 shows a comparison of de-acetylated, dilute acid pretreated cornstover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T.maratima β-glucosidase) or a commercial enzyme preparation (Ctec2).

FIG. 15 shows a comparison of de-acetylated, dilute acid pretreated cornstover conversion by a CelA mixture (17 mg/g CelA; 2 mg/g E1; 1 mg/g T.maratima β-glucosidase) with or without a reducing agent (1 mMcysteine).

DETAILED DESCRIPTION

Disclosed herein are enzymes useful for the hydrolysis of cellulose andthe conversion of biomass. While single enzymes disclosed herein digestcellulose and biomass, various combinations of the enzymes showsynergistic activities on the substrates. Methods of degrading celluloseand biomass using enzymes and cocktails of enzymes are also disclosed.

Enzymatic conversion of biomass is currently performed using mixtures ofmesophillic enzymes derived from fungi such as T. reesei. These mixturesutilize GH 6 and 7 cellulases to perform most of the cellulosehydrolysis work. An alternative approach is to utilize enzymes fromhyperthermophillic bacterial organisms that contain no GH 7 enzymes foreither simultaneous saccharification and fermentation (SSF) or singlestep direct microbial conversion of biomass. These hyperthermophillicsystems utilize a combination of GH 9 and GH 48 enzymes to deconstructcellulose and can operate at extremely high temperatures.

One such system is the cellulase system from Caldicellulosiruptorbescii, which includes the multidomain (GH 9/GH 48) enzyme CelA. Asdiscussed in detail below, the addition of a thermostable betaglucosidase (β-glucosidase) (e.g., from Thermotoga maritima) and/or athermostable endoglucanase (e.g., from Acidothermus cellulolyticus) topurified enzyme preparations of CelA or to C. bescii culture brothssynergistically improves digestion of cellulose and biomass. Theaddition of these enzymes to an existing enzyme cocktail or a hostorganism used to produce an enzyme mixture may also significantlyimprove the performance of these enzyme mixtures, thus improving overallconversion yields, lowering the total enzyme concentrations required toreach a specified level of conversion or reducing the total timerequired to reach a specified level of conversion.

Without being bound by any particular theory, thermostableendoglucanases and beta-glucosidases may synergistically enhance thefunction of C. bescii cellulases such as CelA by increasing the numberof reducing ends accessible to the CelA enzyme and relieving end productinhibition effects. As used herein, “thermostable” refers to enzymesthat exhibit significant activity at elevated temperatures (e.g., over70° C.) for several days.

Thermostable endoglucanases such as E1 from A. cellulolyticus combinedwith thermostable β-glucosidases such as those from T. maritima maysynergistically improve the activity of GH 9/GH 48 enzymes such as CelAenzymes or cocktails containing CelA enzymes acting on biomass and theoverall extent of conversion of biomass by the combined enzymes. Theenhanced extent of glucan conversion exhibited by this enzymecombination indicates that there is synergy between the A.cellulolyticus E1 endoglucanase and T. maritima β-glucosidase with theC. bescii CelA enzyme, as well as the whole C. bescii culture broth.

The Caldicellulosiruptor species C. bescii presents several key featuresthat make it an attractive candidate as a consolidated bioprocessing(CBP) microbe. For example, C. bescii grows well on crystallinecellulose as well as unpretreated substrates such as switchgrass andpoplar at 75° C. Other species of the genus Caldicellulosiruptor includeC. obsidiansis, C. kronotskiensis, C. hydrothermalis, C. owensensis, C.saccharolyticus, C. lactoaceticus, C. acetigenus, and C. kristjanssonii.

CelA is a complex enzyme containing an N-terminal GH9endo-beta-1,4-glucanase, three family 3 carbohydrate binding modules(CBMIII), and a C-terminal GH48 exo-beta-1,4-glucanase. CelA is capableof withstanding temperatures over 90° C. and has an optimal activity atthe 85-90° C. range making it a good candidate for an industrial processwhere higher temperatures are desired and where costly steps afterpretreatment can be avoided. CelA also combines the strengths of achain-end forming endoglucanase and an efficient cellobiohydrolase onthe same protein. This combination allows CelA to be efficient on highlycrystalline cellulose substrates. Also, it outperforms one of the mostefficient fungal combinations of Exo/Endo-glucanases on avicel even atthe temperature of 60° C., far from ideal for CelA. Finally, CelAexhibits hydrolytic activity on xylan—an attractive feature from aprocess standpoint where biomass feedstocks used are rich in xylan.

β-glucosidases are a family of exocellulase enzymes that catalyze thecleavage of β(1-4) linkages in substrates such as cellobiose, resultingin the release of glucose. The β-glucosidase from T. maritima (whosesequence is set forth in FIG. 2) is provided as a specific example, butother β-glucosidases may be suitable for use in the enzyme cocktails andmethods described herein.

Suitable thermostable β-glucosidases include those derived from bacteriaof the genus Thermotoga, including the species T. maritima. Otherspecies of the genus Thermotoga include T. elfii, T. hypogeal, T.lettingae, T. naphthophila, T. neapolitana, T. petrophila, T.subterranean, and T. thermarum.

Endoglucanases suitable for use in the cocktails and methods includethermostable endoglucanases from organisms of the genus Acidothermus,including A. cellulolyticus, and the E1 endoglucanase from A.cellulolyticus (the sequence of which is presented in FIG. 3).

The components of enzyme cocktails may be varied depending on the natureof the substrate being degraded and the pretreatment protocol applied tothe substrate. Exemplary enzyme cocktails may comprise, by weight,30-95% of a thermostable GH 9/GH 48 enzyme such as CelA, 5-25% of athermostable β-glucosidase, 5-40% of a thermostable endoglucanase suchas E1, and 1-20% of additional enzymes such as xylanases (e.g., thethermostable XynA from A. cellulolyticus) or β-xylosidases. Accessoryenzymes may also be included at relatively small percentages of theenzyme cocktail. A thermostable GH 9/GH 48 enzyme such as CelA may becomprise at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95% of the enzyme cocktail. A thermostableβ-glucosidase may comprise at least about 5%, 10%, 15%, 20%, or 25% ofthe enzyme cocktail. A thermostable endoglucanase such as E1 maycomprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% of theenzyme cocktail. A thermostable xylanase or β-xylosidase may comprise atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19% or 20% of the enzyme cocktail. Additionalaccessory enzymes may comprise at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9% or 10% of the enzyme cocktail.

The enzyme cocktails exhibit surprisingly improved cellulase activitieswhen compared to the individual enzyme activities or the additive effectof each enzyme. The term “improved activity” refers to an increased rateof conversion of a cellulosic substrate or a specific component thereof.Relative activities can be determined using conventional assays,including those discussed in the Examples below. Additional assayssuitable for determining cellulase activity include hydrolysis assays onindustrially relevant cellulose-containing substrates such as pretreatedcorn stover. Hydrolysis assays on crystalline cellulose or amorphouscellulose or on small molecule fluorescent reporters may also be used todetermine cellulase activity. In certain embodiments, cellulase activityis expressed as the amount of time or enzyme concentration needed toreach a certain percentage (e.g., 80%) of cellulose conversion tosugars.

Enzymes described herein may be used as purified recombinant enzyme oras culture broths from cells that naturally produce the enzyme or thathave been engineered to produce the enzyme. In certain embodiments,enzyme cocktails may achieve cellulose conversions to sugars (as apercentage of the total cellulose in the original substrate) rangingfrom 50% to 100%, 70% to 100%, or 90% to 100%. In some embodiments, thecellulose conversion exhibited by the enzyme cocktail may be at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

Methods for degrading cellulose and materials containing cellulose usingthe enzymes and enzyme cocktails are also provided herein. For example,the enzyme cocktails may be used in compositions to help degrade (e.g.,by liquefaction) a variety of cellulose products (e.g., paper, cotton,etc.) in landfills. The enzyme cocktails may also be used to enhance thecleaning ability of detergents, function as a softening agent or improvethe feel of cotton fabrics (e.g., stone washing or biopolishing) or infeed compositions.

Cellulose containing materials may also be degraded to sugars using theenzymes. Ethanol may be subsequently produced from the fermentation ofsugars derived from the cellulosic materials. Exemplarycellulose-containing materials include bioenergy crops, agriculturalresidues, municipal solid waste, industrial solid waste, sludge frompaper manufacture, yard waste, wood and forestry waste. Examples ofbiomass include, but are not limited to, corn grain, corn cobs, cropresidues such as corn husks, corn stover, corn fiber, grasses, wheat,wheat straw, barley, barley straw, hay, rice straw, switchgrass, wastepaper, sugar cane bagasse, sorghum, soy, components obtained frommilling of grains, trees, branches, roots, leaves, wood (e.g., poplar)chips, sawdust, shrubs and bushes, vegetables, fruits, flowers andanimal manure.

Biofuels such as ethanol may be produced by saccharification andfermentation of lignocellulosic biomass such as trees, herbaceousplants, municipal solid waste and agricultural and forestry residues.Typically, saccharification is carried out by contacting thelignocellulosic biomass with an enzyme cocktail that includes one ormore of the enzymes described herein. Such enzyme cocktails may alsocontain one or more endoglucanases (such as the Family 5 endoglucanaseE1 from Acidothermus cellulolyticus) or one or more β-glucosidases(e.g., a β-glucosidase from A. niger) to optimize hydrolysis of thelignocelluloses. Additional suitable endoglucanases include EGI, EGII,EGIII, EGIV, EGV or Cel7B (e.g., Cel7B from T. reesei). Enzyme cocktailsmay also include accessory enzymes such as hemicellulases, pectinases,oxidative enzymes, and the like.

Enzymes with the ability to degrade carbohydrate-containing materials,such as cellulases with endoglucanase activity, exoglucanase activity,or β-glucosidase activity, or hemicellulases with endoxylanase activity,exoxylanase activity, or β-xylosidase activity may be included in enzymecocktails. Examples include enzymes that possess cellobiohydrolase,α-glucosidase, xylanase, β-xylosidase, α-galactosidase, β-galactosidase,α-amylase, glucoamylases, arabinofuranosidase, mannanase, β-mannosidase,pectinase, acetyl xylan esterase, acetyl mannan esterase, ferulic acidesterase, coumaric acid esterase, pectin methyl esterase, laminarinase,xyloglucanase, galactanase, glucoamylase, pectate lyase, chitinase,exo-β-D-glucosaminidase, cellobiose dehydrogenase, ligninase, amylase,glucuronidase, ferulic acid esterase, pectin methyl esterase, arabinase,lipase, glucosidase or glucomannanase activities.

A lignocellulosic biomass or other cellulosic feedstock may be subjectedto pretreatment at an elevated temperature in the presence of a diluteacid, concentrated acid or dilute alkali solution for a time sufficientto at least partially hydrolyze the hemicellulose components beforeadding the enzyme cocktail. Additional suitable pretreatment regimensinclude ammonia fiber expansion (AFEX), treatment with hot water orsteam, or lime pretreatment. A lignocellulosic biomass or othercellulosic feedstock may also be de-acetylated before or after thepretreatment regimens listed above.

Lignocellulosic biomass and other cellulose containing materials arecontacted with enzymes at a concentration and a temperature for a timesufficient to achieve the desired amount of cellulose degradation. Theenzymes and cocktails disclosed herein may be used at any temperature,but are well suited for higher temperature digestions. For example, theenzymes or cocktails may be used at temperatures ranging from about 50°C. to about 60° C., from about 60° C. to about 70° C., from about 70° C.to about 80° C., from about 80° C. to about 90° C., from about 90° C. toabout 100° C., from about 50° C. to about 100° C., from about 60° C. toabout 90° C., from about 70° C. to about 85° C., or from about 80° C. toabout 85° C. Exemplary temperatures include 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89 and 90° C.

Suitable times for cellulose degradation range from a few hours toseveral days, and may be selected to achieve a desired amount ofdegradation. Exemplary digestion times include 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 hours; and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5,14, 14.5 or 15 days. In some embodiments, digestion times may be one ormore weeks.

A reducing agent may also be added to the digestion mixture to improvethe cellulose degradation by the enzyme mixture. Exemplary reducingagents include cysteine and dithiothreitol. Reducing agents may be addedat concentrations ranging from 1 nM to 100 mM (e.g., 1 mM).

Separate saccharification and fermentation is a process wherebycellulose present in biomass is converted to glucose that issubsequently converted to ethanol by yeast or bacteria strains.Simultaneous saccharification and fermentation is a process wherebycellulose present in biomass is converted to glucose and, at the sametime and in the same reactor, converted into ethanol by yeast orbacteria strains. Enzyme cocktails may be added to the biomass prior toor at the same time as the addition of a fermentative organism.

The resulting products after cellulose degradation may also be convertedto products other than ethanol. Examples include conversion to higheralcohols, hydrocarbons, or other advanced fuels via biological orchemical pathways, or combination thereof.

“Nucleic acid” or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides.This includes single- and double-stranded molecules (i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

Nucleic acids referred to herein as “isolated” are nucleic acids thathave been removed from their natural milieu or separated away from thenucleic acids of the genomic DNA or cellular RNA of their source oforigin (e.g., as it exists in cells or in a mixture of nucleic acidssuch as a library), and may have undergone further processing. Isolatednucleic acids include nucleic acids obtained by methods describedherein, similar methods or other suitable methods, including essentiallypure nucleic acids, nucleic acids produced by chemical synthesis, bycombinations of biological and chemical methods, and recombinant nucleicacids that are isolated.

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures that rely upon a methodof artificial replication, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. Recombinantnucleic acids also include those that result from recombination eventsthat occur through the natural mechanisms of cells, but are selected forafter the introduction to the cells of nucleic acids designed to allowor make probable a desired recombination event. Portions of isolatednucleic acids that code for polypeptides having a certain function canbe identified and isolated by, for example, the method disclosed in U.S.Pat. No. 4,952,501.

An isolated nucleic acid molecule can be isolated from its naturalsource or produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning) or chemical synthesis.Isolated nucleic acid molecules can include, for example, genes, naturalallelic variants of genes, coding regions or portions thereof, andcoding and/or regulatory regions modified by nucleotide insertions,deletions, substitutions, and/or inversions in a manner such that themodifications do not substantially interfere with the nucleic acidmolecule's ability to encode a polypeptide or to form stable hybridsunder stringent conditions with natural gene isolates. An isolatednucleic acid molecule can include degeneracies. As used herein,nucleotide degeneracy refers to the phenomenon that one amino acid canbe encoded by different nucleotide codons. Thus, the nucleic acidsequence of a nucleic acid molecule that encodes a protein orpolypeptide can vary due to degeneracies.

Unless so specified, a nucleic acid molecule is not required to encode aprotein having protein activity. A nucleic acid molecule can encode atruncated, mutated or inactive protein, for example. In addition,nucleic acid molecules may also be useful as probes and primers for theidentification, isolation and/or purification of other nucleic acidmolecules, independent of a protein-encoding function.

Suitable nucleic acids include fragments or variants that encode afunctional enzyme. For example, a fragment can comprise the minimumnucleotides required to encode a functional cellulase. Nucleic acidvariants include nucleic acids with one or more nucleotide additions,deletions, substitutions, including transitions and transversions,insertion, or modifications (e.g., via RNA or DNA analogs). Alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence.

In certain embodiments, a nucleic acid may be identical to the sequencesrepresented in FIGS. 1-3. In other embodiments, the nucleic acids may beleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequencesrepresented in FIG. 1-3, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequences represented in FIGS. 1-3. Sequence identity calculations canbe performed using computer programs, hybridization methods, orcalculations. Exemplary computer program methods to determine identityand similarity between two sequences include, but are not limited to,the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLASTprograms are publicly available from NCBI and other sources. Forexample, nucleotide sequence identity can be determined by comparingquery sequences to sequences in publicly available sequence databases(NCBI) using the BLASTN2 algorithm.

Embodiments of the nucleic acids include those that encode thepolypeptides that functions as cellulases or functional equivalentsthereof. The amino acid sequences of exemplary enzymes are depicted inFIGS. 1-3. A functional equivalent includes fragments or variants ofthese that exhibit the ability to function as a cellulase. As a resultof the degeneracy of the genetic code, many nucleic acid sequences canencode a polypeptide having, for example, the amino acid sequence Shownin FIGS. 1-3. Such functionally equivalent variants are contemplatedherein.

Altered or variant nucleic acids can be produced by one of skill in theart using the sequence data illustrated herein and standard techniquesknown in the art. Variant nucleic acids may be detected and isolated byhybridization under high stringency conditions or moderate stringencyconditions, for example, which are chosen to prevent hybridization ofnucleic acids having non-complementary sequences. “Stringencyconditions” for hybridizations is a term of art that refers to theconditions of temperature and buffer concentration that permithybridization of a particular nucleic acid to another nucleic acid inwhich the first nucleic acid may be perfectly complementary to thesecond, or the first and second may share some degree of complementaritythat is less than perfect.

Nucleic acids may be derived from a variety of sources including DNA,cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Suchsequences may comprise genomic DNA, which may or may not includenaturally occurring introns. Moreover, such genomic DNA may be obtainedin association with promoter regions or poly (A) sequences. Thesequences, genomic DNA, or cDNA may be obtained in any of several ways.Genomic DNA can be extracted and purified from suitable cells by meanswell known in the art. Alternatively, mRNA can be isolated from a celland used to produce cDNA by reverse transcription or other means.

Also disclosed herein are recombinant vectors, including expressionvectors, containing nucleic acids encoding enzymes. A “recombinantvector” is a nucleic acid molecule that is used as a tool formanipulating a nucleic acid sequence of choice or for introducing such anucleic acid sequence into a host cell. A recombinant vector may besuitable for use in cloning, sequencing, or otherwise manipulating thenucleic acid sequence of choice, such as by expressing or delivering thenucleic acid sequence of choice into a host cell to form a recombinantcell. Such a vector typically contains heterologous nucleic acidsequences not naturally found adjacent to a nucleic acid sequence ofchoice, although the vector can also contain regulatory nucleic acidsequences (e.g., promoters, untranslated regions) that are naturallyfound adjacent to the nucleic acid sequences of choice or that areuseful for expression of the nucleic acid molecules.

The nucleic acids described herein may be used in methods for productionof enzymes and enzyme cocktails through incorporation into cells,tissues, or organisms. In some embodiments, a nucleic acid may beincorporated into a vector for expression in suitable host cells. Thevector may then be introduced into one or more host cells by any methodknown in the art. One method to produce an encoded protein includestransforming a host cell with one or more recombinant nucleic acids(such as expression vectors) to form a recombinant cell. The term“transformation” is generally used herein to refer to any method bywhich an exogenous nucleic acid molecule (i.e., a recombinant nucleicacid molecule) can be inserted into a cell, but can be usedinterchangeably with the term “transfection.”

Non-limiting examples of suitable host cells include cells frommicroorganisms such as bacteria, yeast, fungi, and filamentous fungi.Exemplary microorganisms include, but are not limited to, bacteria suchas strains of Bacillus brevis, Bacillus megaterium, Bacillus subtilis,Caulobacter crescentus, and Escherichia coli (e.g., BL21 and K12);filamentous fungi from the genera Trichoderma (e.g., T. reesei, T.viride, T. koningii, or T. harzianum), Penicillium (e.g., P.funiculosum), Humicola (e.g., H. insolens), Chrysosporium (e.g., C.lucknowense), Gliocladium, Aspergillus (e.g., A. niger, A. nidulans, A.awamori, or A. aculeatus), Fusarium, Neurospora, Hypocrea (e.g., H.jecorina), and Emericella; and yeasts from the genera Saccharomyces(e.g., S. cerevisiae), Pichia (e.g., P. pastoris), or Kluyveromyces(e.g., K. lactis). Cells from plants such as Arabidopsis, barley,citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switchgrass, alfalfa, miscanthus, and trees such as hardwoods and softwoodsare also contemplated herein as host cells.

Host cells can be transformed, transfected, or infected as appropriateby any suitable method including electroporation, calcium chloride-,lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-,DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection,microinjection, microprojectile bombardment, phage infection, viralinfection, or other established methods. Alternatively, vectorscontaining the nucleic acids of interest can be transcribed in vitro,and the resulting RNA introduced into the host cell by well-knownmethods, for example, by injection. Exemplary embodiments include a hostcell or population of cells expressing one or more nucleic acidmolecules or expression vectors described herein (for example, agenetically modified microorganism). The cells into which nucleic acidshave been introduced as described above also include the progeny of suchcells.

Vectors may be introduced into host cells such as those from filamentousfungi by direct transformation, in which DNA is mixed with the cells andtaken up without any additional manipulation, by conjugation,electroporation, or other means known in the art. Expression vectors maybe expressed by filamentous fungi or other host cells episomally or thegene of interest may be inserted into the chromosome of the host cell toproduce cells that stably express the gene with or without the need forselective pressure. For example, expression cassettes may be targeted toneutral chromosomal sites by recombination.

Host cells carrying an expression vector (i.e., transformants or clones)may be selected using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule.In prokaryotic hosts, the transformant may be selected, for example, byresistance to ampicillin, tetracycline or other antibiotics. Productionof a particular product based on temperature sensitivity may also serveas an appropriate marker.

Host cells may be cultured in an appropriate fermentation medium. Anappropriate, or effective, fermentation medium refers to any medium inwhich a host cell, including a genetically modified microorganism, whencultured, is capable of growing or expressing the polypeptides describedherein. Such a medium is typically an aqueous medium comprisingassimilable carbon, nitrogen and phosphate sources, but can also includeappropriate salts, minerals, metals and other nutrients. Microorganismsand other cells can be cultured in conventional fermentation bioreactorsand by any fermentation process, including batch, fed-batch, cellrecycle, and continuous fermentation. The pH of the fermentation mediumis regulated to a pH suitable for growth of the particular organism.Culture media and conditions for various host cells are known in theart. A wide range of media for culturing filamentous fungi, for example,are available from ATCC. Exemplary culture/fermentation conditions andreagents are provided in the Examples that follow.

The nucleic acid molecules described herein encode the enzymes withamino acid sequences such as those represented by FIGS. 1-3. As usedherein, the terms “protein” and “polypeptide” are synonymous. “Peptides”are defined as fragments or portions of polypeptides, preferablyfragments or portions having at least one functional activity as thecomplete polypeptide sequence. “Isolated” proteins or polypeptides areproteins or polypeptides purified to a state beyond that in which theyexist in cells. In certain embodiments, they may be at least 10% pure;in others, they may be substantially purified to 80% or 90% purity orgreater. Isolated proteins or polypeptides include essentially pureproteins or polypeptides, proteins or polypeptides produced by chemicalsynthesis or by combinations of biological and chemical methods, andrecombinant proteins or polypeptides that are isolated. Proteins orpolypeptides referred to herein as “recombinant” are proteins orpolypeptides produced by the expression of recombinant nucleic acids.

Proteins or polypeptides encoded by nucleic acids as well as functionalportions or variants thereof are also described herein. Polypeptidesequences may be identical to the amino acid sequences presented inFIGS. 1-3, or may include up to a certain integer number of amino acidalterations. Such protein or polypeptide variants retain functionalityas cellulases, and include mutants differing by the addition, deletionor substitution of one or more amino acid residues, or modifiedpolypeptides and mutants comprising one or more modified residues. Thevariant may have one or more conservative changes, wherein a substitutedamino acid has similar structural or chemical properties (e.g.,replacement of leucine with isoleucine). Alterations may occur at theamino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence.

In certain embodiments, the polypeptides may be at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequences set forth inFIGS. 1-3 and possess enzymatic function. Percent sequence identity canbe calculated using computer programs (such as the BLASTP and TBLASTNprograms publicly available from NCBI and other sources) or directsequence comparison. Polypeptide variants can be produced usingtechniques known in the art including direct modifications to isolatedpolypeptides, direct synthesis, or modifications to the nucleic acidsequence encoding the polypeptide using, for example, recombinant DNAtechniques.

Polypeptides may be retrieved, obtained, or used in “substantially pure”form, a purity that allows for the effective use of the protein in anymethod described herein or known in the art. For a protein to be mostuseful in any of the methods described herein or in any method utilizingenzymes of the types described herein, it is most often substantiallyfree of contaminants, other proteins and/or chemicals that mightinterfere or that would interfere with its use in the method (e.g., thatmight interfere with enzyme activity), or that at least would beundesirable for inclusion with a protein.

EXAMPLES Example 1 Bacterial Strains and Growth Conditions

C. bescii DSM 6725 cells were grown at 75° C. under anaerobic conditionson Avicel. Non-avicel bound proteins were pre-affinity purified on aphenyl sepharose column.

Example 2 Purification of CelA

The CelA holoenzyme was initially purified with anion exchangechromatography using a Source 15Q column (GE Healthcare, Piscataway,N.J.). For this chromatography, buffer A was 20 mM Tris, pH 6.8 andbuffer B was 20 mM Tris, pH 6.8, with 1 M NaCl. The resulting fractionwas then further purified using a source 15PHE hydrophobic interactioncolumn (GE Healthcare, Piscataway, N.J.), with buffer A and buffer C, 20mM acetate, pH 5.0, with 1 M ammonium sulfate. Relevant fractions werethen subjected to size exclusion chromatography using a Sephacryl 300column (GE Healthcare, Piscataway, N.J.) and eluted with buffer D, 20 mMacetate, pH 5.0, and 100 mM NaCl. The relevant CelA containing fractionswere further purified with anion exchange chromatography using a Source15Q column (GE Healthcare, Piscataway, N.J.) and buffer F, 20 mMTris-HCL, pH 8.0, and buffer G, 20 mM Tris-HCL, pH 8.0, with 1 M NaCl.The relevant CelA containing fractions were further purified withhydrophobic interaction chromatography using a Source 15ISO column (GEHealthcare, Piscataway, N.J.) with buffer A (20 mM Tris-HCL, pH 8.0) andbuffer B (20 mM Tris-HCL, pH 8.0 with 1 M ammonium sulfate. TheCelA-containing fractions were once again subjected to size exclusionchromatography using a Sephacryl 300 column (GE Healthcare, Piscataway,N.J.) and eluted with buffer D, 20 mM acetate, pH 5.0 and 100 mM NaCl.The purified fusion proteins were concentrated with a Vivaspin 10Kconcentrator (Vivaproducts, Littleton, Mass.), and the proteinconcentration was determined with a Pierce BCA protein assay (Pierce,Rockford, Ill.).

Example 3 Expression of CelA GH48 and GH9 Modules

CelA CBM3-GH48 construct was overexpressed with N-terminal his-tag in E.coli. It was amplified by primers ACACCGGCTAGCAGCAGCACACCTGTAGCAGG (SEQID NO:7) and TAGCTTCTCGAGTTATTGATTGCCAAACAGTA (SEQ ID NO:8) (therestriction sites are underlined), and the template of the genomic DNAof C. bescii was employed. The PCR fragment was inserted into theplasmid of pET28b (Novagen, Madison, Wis.), and was overexpressed in theE. coli BL21(DE3) strain (Stratagene, La Jolla, Calif.) at 37° C. withaddition of 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

The GH9 construct from CelA was overexpressed with N-terminal his-tag inE. coli. It was amplified by primers of ATGCTAGCTAGCGGTTCGTTTAACTATGGGGA(SEQ ID NO:9) and GTCGTTCTCGAGTCATTCAATAGCTTTGAAATCTG (SEQ ID NO:10)(the restriction sites are underlined), and the template of the genomicDNA of C. bescii was employed. The PCR fragment was inserted into theplasmid of pET28b (Novagen, Madison, Wis.), and was overexpressed in theE. coli BL21(DE3) strain (Stratagene, La Jolla, Calif.) at 37° C. withaddition of 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Example 4 Purification of CelA GH48 and GH9 Modules

The CelA GH48 component was first desalted into 20 mM acetic acid, pH5.5, and then ammonium sulfate was added to a concentration of 2 M. Thisfraction was subjected to hydrophobic interaction chromatography using aSource 15PHE column (GE Healthcare, Piscataway, N.J.). For thischromatography, buffer A was 20 mM acetic acid, pH 5.5, and with 2 Mammonium sulphate, buffer B was 20 mM acetic acid, pH 5.5. The resultingpeak was then purified using a source 15Q anion interaction column (GEHealthcare, Piscataway, N.J.), with buffer A (20 mM Tris, pH 6.8) andbuffer C (20 mM Tris, pH 6.8, with 1 M NaCl. Finally, CelA-GH48 wasseparated from minor impurities by size exclusion chromatography usingHiLoad Superdex 75 (26/60) (GE Healthcare, Piscataway, N.J.) in buffer H(20 mM acetate, pH 5.0, with 100 mM NaCl). The purified protein wasconcentrated with a Vivaspin 5K concentrator (Vivaproducts, Littleton,Mass.), and the concentration determined using the Pierce BCA assay(Pierce, Rockford, Ill.).

The C. bescii GH9 module was further purified by first dialyzing ittwice against 1.5 L of buffer A (20 mM acetic acid, pH 5.5, 1 mM EDTAwith 1 mM DTT) followed by cation exchange chromatography using a Source15S column (GE Healthcare, Piscataway, N.J.). For this chromatography,buffer A and buffer B with additional 1 M NaCl were used. Finally, minorimpurities were removed by size exclusion chromatography using HiLoadSuperdex 75 (26/60) (GE Healthcare, Piscataway, N.J.) in 20 mM Tris-HCl,pH 7.0, containing 100 mM NaCl, 5 mM CaCl₂, 1 mM EDTA, and 1 mM sodiumazide. The purified protein solution was concentrated with a Vivaspin 5Kconcentrator (Vivaproducts, Littleton, Mass.), and its concentration wasmeasured using a NanoDrop UV spectrophotometer (NanoDrop, Wilmington,Del.).

Example 5 Substrates and Biomass Samples

Avicel (ph101), corn stover, and switchgrass samples were used toevaluate the cellulolytic efficiency of CelA, raw C. bescii broth, andenzyme cocktails. The biomass samples were submitted to several types ofpretreatments with increasing severity including diluted acid andammonia fiber explosion (AFEX) pretreatments for both corn stover andswitchgrass; and alkaline peroxide pretreatment for corn stover. Thedetails of each pretreatment condition are described in Table 1.

TABLE 1 Pretreatement conditions Temp Time Compositional analysis (%)Catalyst (° C.) (min) Glucan Xylan Galactan Arabinan Mannan Lignin AshProtein Dilute acid % 0.5 150 20 39.73 21.18 3 24.3 7.06 pretreated corn₄SO₂H 1.33 ND 1.56 stover Alkaline % 2.5 65 60 59 28 0 1.9 0 6.2 1 NDperoxide ₂O₂H pretreated corn stover Native NA NA NA 30.2 21.3 ND 3.15.3 17.4 6.4 2.3 Switchgrass Dilute Acid % 5 190 1 42.59 5.48 0.3 021.52 6.09 Pretreated ₄SO₂H 0.47 ND Switchgrass AFEX g/g 1.52 150 3030.2 21.3 ND 3.1 5.3 17.4 6.4 2.3 pretreated biomass Switchgrass Avicelph101 NA NA NA 94.94 1.48 0.12 0.31 ND 0.54 0.1 ND De-acetylated, 63.915.01 0.63 0.82 23.93 4.07 dilute acid pretreated corn stover

To provide a basis for the maximum theoretical sugar yield achievablefrom each substrate during enzymatic hydrolysis, portions of each of thepretreated solids samples were dried and subjected to the standardtwo-stage sulfuric acid hydrolysis method for determining structuralcarbohydrates in lignocelluloses as described by Sluiter and coworkers(NREL Technical Report: NREL/TP-510-42623 (2006)). In this method, thecarbohydrate content of each pretreated sample is calculated from thecarbohydrates released.

Example 6 Enzyme Activity Assays

Enzyme activities were determined at 60° C., 75° C., and 85° C. at anenzyme concentration of 15 or 20 mg protein per g glucan. Digestionassays using fungal enzymes were performed at 50° C. and an enzymeconcentration of 12 mg/g of Cbh1 from T. reesei and 3 mg/g of E1 from A.cellulolyticus. Both bacterial and fungal digestion assays were done in20 mM acetate, pH 5.5, containing 10 mM CaCl₂, and 100 mM NaCl withcontinuous mixing.

Digestions were run continuously for seven days and sugar release wasmonitored. Samples were taken at various time points, enzymes wereinactivated by boiling for 15 minutes and samples were then filteredthrough 0.45 um Acrodisc syringe filters and analyzed for Glucose,Xylose, and Cellobiose by HPLC. Samples were injected at 20 μL and runon an Agilent 1100 HPLC system equipped with a BioRad Aminex HPX-87H 300mm×7.8 mm column heated to 55° C. A constant flow of 0.6 mL/min was usedwith 0.1 M H₂SO₄ in water as the mobile phase to give separation of theanalytes. Glucose, xylose, and cellobiose were quantified againstindependent standard curves. All experiments were performed intriplicate and the resulting extents of conversion are shown as percentglucan converted.

Additionally, CelA and its two recombinant catalytic domains, GH48 andGH9, were tested for activity on 5 mMpara-nitrophenol-β-D-xylopyranoside (PnP-X) andpara-nitrophenol-β-D-cellobioside (PnP-C) in 20 mM acetate buffer, pH5.5, 50 mM NaCl at 75° C. The incubation was performed for one hour andthen quenched with 50 μl of 1 M calcium carbonate. Absorbance was readat 405 nm.

Example 7 Crystallization

Screening for crystals was done with sitting drop vapor diffusion usinga 96-well plate and Crystal Screen HT, PEG ion HT and Grid Screen SaltHT from Hampton Research (Aliso Viejo, Calif.). 50 μL of well solutionwith drops containing 1 μL of well solution and 1 μL of protein solutionwere used for screening and a 24-well hanging drop vapor diffusion setupwith 1 ml of well solution and drops containing 1 μL of well solutionand 1 μL of protein solution was used for optimization ofcrystallization conditions. The GH9 protein solution contained 9.5 mg/mLof protein, 20 mM Tris pH 7, 100 mM NaCl, 5 mM CaCl₂, 1 mM EDTA and 1 mMNa azide. The best crystals for the unliganded GH9 formed in 0.1 M Nacacodylate pH 6.4, 1.8 M ammonium sulfate and 10% dioxane using a 24well optimization plate. Before flash freezing, the crystal was soakedin a 2 μL cryo solution drop with 10% (v/v) ethylene glycol and 10%(v/v) glycerol in the well solution and incubated for 5 seconds. Thecrystals of GH9 with cellobiose were obtained using other crystals fromthe same condition with an excess amount of cellobiose powder in thecryo solution drop and by incubating for 30 seconds. The GH48 proteinsolution contained 1.8 mg/mL of protein in 20 mM acetic acid pH 5, with100 mM NaCl. Crystals were grown in 0.1 M tri-sodium citrate, pH 5.8,20% (w/v) PEG 4000 and 20% (v/v) 2-propanol using a 24 well optimizationplate. Before flash freezing, the crystal was incubated for 5 seconds ina 2 μL cryo solution drop containing 10% (v/v) ethylene glycol and 10%(v/v) glycerol in the well solution.

Example 8 X-Ray Diffraction, Structure Determination, and StructureAnalysis

Before data collection, all crystals were flash-frozen in a coldnitrogen gas stream at 100 K. Data collection was performed using aBruker X8 MicroStar X-Ray generator with Helios mirrors and BrukerPlatinum 135 CCD detector. Data was indexed and processed with theBruker Suite of programs version 2008.1-0 (Bruker AXS, Madison, Wis.).Intensities were converted into structure factors and 5% of thereflections were flagged for Rfree calculations using programs F2MTZ,Truncate, CAD and Unique from the CCP4 package of programs. The GH9structures were solved using molecular replacement program Molrep withPDB entry 1KSD as a model. The GH48 structure was solved using MrBumpwith PDB entry 1G9G as a model. For all three structures, ARP/wARPversion 7.0 and Coot version 0.6.2 was used for multiple cycles ofautomatic and manual model building. Further refinement and manualcorrection was performed using REFMACS version 5.6.0117 and Coot. Theresulting structures have been deposited to the Protein Data Bank withPDB codes 4DOD (GH9), 4DOE (GH9-CB) and 4EL8 (GH48).

Programs Coot, PyMOL and ICM (molsoft) were used for comparing andanalyzing structures. Ramachandran plot statistics were calculated usingMolprobity and root mean square deviations (rmsd) of bond lengths andangles were calculated from ideal values of Engh and Huberstereochemical parameters. Wilson B-factor was calculated usingCTRUNCATE version 1.0.11. Structural similarity searches were done usingpair wise secondary structure matching by PDBefold.

Example 9 Enzymatic Activity on Model Substrates

The cellulolytic performance of purified CelA, isolated from the C.bescii enzyme broth, was examined at 60° C., 75° C., and 85° C. Toremove any variability in the enzymatic digestion of biomass substratessuch as differences in glucan content or pretreatments, the firstenzymatic assays were performed with the model cellulose avicel PH-101.The percentage of glucan released over a seven-day digestion is shown inFIG. 4. CelA exhibited the most activity at the highest temperaturetested, 85° C. This single enzyme can convert more than 60% of mostlycrystalline cellulose content in 6 days. While lower temperatures impairthe performance of CelA, losing close to 10-15% of its activity forevery 10-degree temperature drop, its activity remains higher than mostsingle cellulases tested in these conditions. Surprisingly, even attemperature of 60° C. where CelA has lost over 40% of its peak activity,it still outperforms a mixture of the fungal Cel7A from T. reesei (Cbh1)and E1 from A. cellulolyticus. At the same mg/g protein loading, CelAalone is about 2 times as effective compared to a current model mixtureof enzymes with the same Exo-endo activity. However, on a molar basis(since CelA is about 4 times larger in terms of molecular weight), thereis about ¼ less CelA enzyme present, making CelA about 8 times as activeas the Cbh1/E1 combination.

The cellulolytic performance of purified CelA, alone or in combinationwith E1 from A. cellulolyticus and/or β-glucosidase from T. maritima, onavicel at 75° C. was also tested. Total enzyme loadings were 15 mgprotein per g glucan. As shown in FIGS. 7, 9 and 11, the addition ofsmall amounts of β-glucosidase (1 mg per g; FIG. 7) or E1 (4 mg per g;FIG. 9) or both (FIG. 11) to CelA surprisingly enhanced the enzymaticactivity of the CelA in a synergistic manner.

Example 10 Enzymatic Activity on Untreated and Pretreated Biomass

The cellulolytic activity of CelA, C. bescii enzyme broth, and enzymecocktails were tested on native switchgrass samples as well as some withvarying pretreatment severity, including dilute acid (DA) and AFEXpretreatments. These enzymatic assays were performed at 75° C. and at atotal protein loading of 15 mg of enzyme per g of feedstock. Under theseconditions, the maximum glucan conversion of CelA peaks at 30-35% (FIG.5A) on native switchgrass and dilute acid pretreated switchgrass. CelAactivity was the lowest for AFEX pretreated switchgrass were the glucanconversion is 20%. Surprisingly, the activity of CelA on biomass issimilar to that of the C. bescii enzyme broth. For comparison with CelA,the commercial enzyme preparation CTec2 was used on DA pretreatedswitchgrass at its optimal temperature of 50° C. The exact enzymecomposition of CTec2 is unknown as it is proprietary; however, themixture likely contains cellulases, hemicellulases, andbeta-glucosidases from modified fungi. CTec2 achieves 63% conversion ofglucans after 168 hours and outperforms CelA and C. bescii broth at thesame enzyme loading of 15 mg/g.

Similarly, the enzymatic activity of CelA was also tested on a diluteacid (DA) and alkaline peroxide (AP) pretreated corn stover at 75° C.but an enzyme loading of 20 mg of enzyme per g of feedstock (FIG. 5B).CelA performs better on AP corn stover, with a conversion of close to50%. The same result is observed for the C. bescii enzyme broth.

The ability of CelA to convert xylan found in native switchgrass at 75°C. was also examined. As shown in FIG. 6, CelA can efficiently convertxylan when it is present in untreated biomass. Levels of xylanconversion approached 60% in these assays, which is much higher thanexpected.

The enzymatic activity of purified CelA, alone or in combination with E1from A. cellulolyticus and/or β-glucosidase from T. maritima, on DApretreated corn stover at 75° C. was also tested. Total enzyme loadingswere 15 mg protein per g glucan. As with activities seen on avicel, theaddition of small amounts of β-glucosidase (1 mg per g; FIG. 8) or E1 (4mg per g; FIG. 10) or both (FIG. 12) to CelA surprisingly enhanced theenzymatic activity of the CelA in a synergistic manner.

Example 11 Activity of CelA and CelA Components on PnP

The xylanase activity of CelA and its two catalytic units GH48 and GH9was examined on para-nitrophenol-β-D-xylopyranoside (PnP-X) andpara-nitrophenol-β-D-cellobioside (PnP-C). The results shown in Table 2indicate that CelA and the GH48 module have activity on both substrateswhereas the GH9 component has activity on cellulose. CelA was also ableto achieve 60% conversion of xylan from native switchgrass, which showsits potential for an industrial process using mild to no pretreatment.

TABLE 2 PnP-X PnP-C CelA holoenzyme +++ ++++ CelA GH48 ++++ ++++ CelAGH9 − +

Example 12 Crystal Structures of the C. bescii CelA GH9 and GH48 Modules

The structure of the unliganded C. bescii CelA GH9 module was refined toa resolution of 1.7 Å with R and Rfree of 0.155 and 0.179, respectively,and one molecule in the asymmetric unit. The GH9 module with cellobiosewas refined to a resolution of 1.56 Å with R and Rfree of 0.148 and0.175, respectively, and one molecule in the asymmetric unit. GH48 had aresolution of 2.45 Å with R and Rfree of 0.195 and 0.258 with onemolecule in the asymmetric unit.

The GH9 module of CelA has an (alpha/alpha)6 barrel fold. The unligandedGH9 structure has one calcium atom, one 1,4-dioxane molecule, 11ethylene glycol molecules, ten glycerol molecules and four sulfates. Theliganded GH9 module has one cellobiose molecule and one cellotriosemolecule bound at the active site and one calcium atom, one 1,4-dioxanemolecule, 26 ethylene glycol molecules, six glycerol molecules and threesulfates bound elsewhere in the structure.

The GH48 module has an (alpha/alpha)6 barrel fold. The structurerevealed one calcium atom, one ethylene glycol and one sulfate. The GH48structure did not contain the CBM3 module that was included before it inthe construct. The CBM3 module may have been cut from the GH48 becauseof degradation during the crystallization trials. The CelF (PDB entry1FCE) numbering was used, starting from residue seven (of CelF) that wasthe first one visible in the electron density. The last three residuesand a loop formed by residues 308 to 310 were not modeled due to weak orno electron density. The 308 to 310 loop was special because it actuallyhad some density where the corresponding residues from CelF (PDB entry1FCE) and CelS (PDB entry 1L1Y) are but the resulting model would be tooinaccurate due to possible other overlapping conformations making thedensity weak and noisy leading to wrong conformations after refinement.

Pairwise secondary-structure matching of structures with at least 70%secondary structure similarity by PDBefold found 19 unique structuralmatches for the GH9 module (liganded structure was used) and 21 matchesfor GH48. Structures similar to CelA GH9 included other GH9s andglucuronyl hydrolases with varying sequence identities with a GH9 fromClostridium thermocellum (PDB entry 2XFG) being most similar (71%sequence identity). CelA GH48 was only similar to the differentstructures of CelF and CelS GH48.

Example 13 Enzymatic Activity, Time of Addition and Reducing Agents

The cellulolytic activities of enzyme cocktails (17 mg/g CelA; 2 mg/gE1; 1 mg/g T. maratima β-glucosidase) were tested on samples ofde-acetylated, dilute acid pretreated corn stover in a horizontal screwreactor. Results shown in FIG. 13 demonstrate that up to 80% conversionof this substrate can be achieved using the enzyme cocktails.

CelA enzyme cocktails may also be added to a reactor at a highertemperature than commercial enzyme preparations. Because large reactorstake time and water/energy to cool, CelA mixtures can create savings intime and energy by being added earlier in the process (e.g., at 80° C.rather than cooling to 50° C.) without loss of enzymatic activity. FIG.14 shows the activity of CelA mixture in comparison to a commercialenzyme cocktail.

FIG. 15 shows the increase in overall conversion when a reducing agentis added to the reaction mixture. The addition of 1 mM cysteine improvedthe conversion by up to 20%.

The Examples discussed above are provided for purposes of illustrationand are not intended to be limiting. Still other embodiments andmodifications are also contemplated.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

We claim:
 1. A method for degrading cellulose, comprising contacting acellulose containing material with an enzyme cocktail comprising arecombinant thermostable enzyme from Caldicellulosiruptor besciicomprising a glycoside hydrolase (GH9) domain and a glycoside hydrolase(GH48) domain and a recombinant thermostable β-glucosidase, wherein atleast 50% of the cellulose in the material is converted to at least onesugar.
 2. The method of claim 1, wherein the thermostable enzymecomprising a GH9 domain and a GH48 domain is Caldicellulosiruptor besciiCelA.
 3. The method of claim 1, wherein the thermostable β-glucosidaseis from a bacterium of the genus Thermotoga.
 4. The method of claim 1,wherein the enzyme cocktail further comprises a thermostableendoglucanase.
 5. The method of claim 4, wherein the thermostableendoglucanase is from a bacterium of the genus Acidothermus.
 6. Themethod of claim 4, wherein the thermostable endoglucanase isAcidothermus cellulolyticus E1.
 7. A method for degradinglignocellulosic biomass, comprising contacting the lignocellulosicbiomass with an enzyme cocktail comprising a recombinant thermostableenzyme from Caldicellulosiruptor bescii comprising a glycoside hydrolase(GH9) domain and a glycoside hydrolase (GH48) domain and a recombinantthermostable β glucosidase, wherein at least 50% of the cellulose in thebiomass is converted to at least one sugar.
 8. The method of claim 7,wherein the thermostable enzyme comprising a GH9 domain and a GH48domain is Caldicellulosiruptor bescii CelA.
 9. The method of claim 7,wherein the thermostable β-glucosidase is from a bacterium of the genusThermotoga.
 10. The method of claim 7, wherein the enzyme cocktailfurther comprises a thermostable endoglucanase.
 11. The method of claim10, wherein the thermostable endoglucanase is from a bacterium of thegenus Acidothermus.
 12. The method of claim 1, wherein the thermostableenzyme comprising a GH9 domain and a GH48 domain comprises at least 30%of the enzyme cocktail.
 13. The method of claim 1, wherein thethermostable enzyme comprising a GH9 domain and a GH48 domain comprisesat least 50% of the enzyme cocktail.
 14. The method of claim 1, whereinthe thermostable enzyme comprising a GH9 domain and a GH48 domain isCaldicellulosiruptor bescii CelA, wherein the thermostable β-glucosidaseis from the bacterium Thermotoga maritima; and wherein the CelAcomprises at least 30% of the enzyme cocktail and the β-glucosidasecomprises at least about 5% of the enzyme cocktail.
 15. The method ofclaim 14, wherein the enzyme cocktail further comprises at least 5% ofthe thermostable endoglucanase Acidothermus cellulolyticus E1.
 16. Amethod for degrading cellulose, comprising contacting a cellulosecontaining material with an enzyme cocktail comprising CelA fromCaldicellulosiruptor bescii and a thermostable β-glucosidase fromThermotoga maritima; wherein the thermostable β-glucosidase comprisesabout 5% of the enzyme cocktail by weight; and wherein at least 50% ofthe cellulose in the material is converted to at least one sugar. 17.The method of claim 16, wherein at least 70% of the cellulose in thematerial is converted to at least one sugar.
 18. The method of claim 16,wherein 50 to 100% of the cellulose in the material is converted to atleast one sugar.
 19. The method of claim 16, wherein 70 to 100% of thecellulose in the material is converted to at least one sugar.